Inducible expression of a degradation-resistant form of p27Kip1 causes growth arrest and apoptosis in breast cancer cells.

The cyclin-dependent kinase (CDK) inhibitor p27(Kip1) (p27) is an important regulator of cell cycle progression controlling the transition from G to S-phase. Low p27 levels or accelerated p27 degradation correlate with excessive cell proliferation and poor prognosis in several forms of cancer. Phosphorylation of p27 at Thr187 by cyclin E-CDK2 is required to initiate the ubiquitination-proteasomal degradation of p27. Protecting p27 from ubiquitin-mediated proteasomal degradation may increase its potential in cancer gene therapy. Here we constructed a non-phosphorylatable, proteolysis-resistant p27 mutant containing a Thr187-to-Ala substitution (T187A) which is not degraded by ubiquitin-mediated proteasome pathway, and compared its effects on cell growth, cell-cycle control, and apoptosis with those of wild-type p27. In muristerone A-inducible cell lines overexpressing wild-type or mutant p27, the p27 mutant was more resistant to proteolysis in vivo and more potent in inducing cell-cycle arrest and other growth-inhibitory effects such as apoptosis. Transduction of p27(T187A) in breast cancer cells with a doxycycline-regulated adenovirus led to greater inhibition of proliferation, more extensive apoptosis, with a markedly reduced protein levels of cyclin E and increased accumulation of cyclin D1, compared with wild-type p27. These findings support the potential effectiveness of a degradation-resistant form of p27 in breast cancer gene therapy.

A comprehensive assessment of p53-responsive genes following adenoviral-p53 gene transfer in Bcl-2-expressing prostate cancer cells.

The p53 protein can induce cell cycle arrest or apoptosis following activation in response to DNA damage. The function of p53 is largely mediated by regulating the expression of downstream target genes. Adenoviral-p53 gene transfer (Ad-p53) is currently being evaluated in clinical trials as a therapeutic intervention. Tumor response is likely to be influenced by context-dependent variables, such as expression of bcl-2. Bcl-2 is upregulated in a variety of neoplasms, and can inhibit p53-dependent apoptosis. It was therefore of interest to use a global genomic strategy to assess gene expression following Ad-p53 gene transfer and to determine if the expression of specific Ad-p53-responsive genes could be modulated in the context of bcl-2 gene deregulation. cDNA arrays were used to identify p53-responsive genes following Ad-p53 gene transfer in control and bcl-2-overexpressing PC3 prostate cancer cells. A total of 40 transcripts were significantly upregulated by Ad-p53 in both control and bcl-2-transfectant PC3 cells. Conversely, 19 transcripts were significantly repressed in both cell lines. These Ad-p53-responsive transcripts included previously identified p53 targets, known genes representing candidate p53 targets, and transcripts identified as expressed sequence tags. A subset of 15 transcripts was differentially modulated by Ad-p53 in the context of bcl-2. Some of these genes were also differentially modulated in LNCaP (wt p53) cells following DNA damage. These results document a number of potential p53 targets and mediators of therapeutically relevant genotoxic stress. The findings further suggest that bcl-2 may inhibit cell death at multiple points downstream of p53 activation.